The invention relates to treatment of xcex11-antitrypsin (xcex11-AT) deficiency in individuals containing a mutant form of xcex11-AT exhibiting symptoms of, or at-risk for, liver damage and/or emphysema. In particular, the invention concerns the use of inhibitors of glucosidase and in some instances, of mannosidase in ameliorating these conditions.
The enzyme xcex11-antitrypsin (xcex11-AT) is important in maintaining the condition of lung tissue by virtue of its ability to inhibit neutrophil elastase. If this elastase inhibitor is lacking in the lungs, lung diseases such as emphysema can develop. A substantial number of individuals are deficient in this important enzyme by virtue of the presence of a mutant form of the glycoprotein, designated xcex11-ATZ, differing from the wild type by a single amino acid substitution. Although xcex11-ATZ this retains approximately 80% of the functional activity of the wild type in inhibiting neutrophil elastase, because it is misfolded and polymerized in the endoplasmic reticulum (ER) of liver cells rather than excreted into the extracellular fluid, it exerts a hepatotoxic effect, especially in infants and children, and is not available in the lungs to carry out its function. There are, however, known pathways for degradation of the mutant xcex11-ATZ in the ERxe2x80x94one involving the sequence of stable binding to calnexin, conjugation of ubiquitin to the cytoplasmic tail of the complexed calnexin and degradation of the resulting complex by the proteasome (Qu, D., et al., J. Biol. Chem. (1996) 271:22791-22795). There is also a ubiquitin-independent proteasomal mechanism (Teckman, J. H., et al., Biochem J. (1986) 236:853-860).
It is apparent that individuals having the genotype which results in the production of the mutant xcex11-ATZ would benefit if the secretion of this mutant form of xcex11-AT could be enhanced, since this mutant form does retain the desired neutrophil elastase inhibition activity. Such individuals would also benefit by any protocol which would result in the enhanced degradation of this mutant form in the ER, thus ameliorating the hepatotoxicity of the misfolded polymer. It has been shown that glucosidase and mannosidase inhibitors inhibit secretion of wild type xcex11-AT (Gross, V., et al, Biochem. J. (1986) 236:853-860). It has also been shown that a different mutant xcex11-AT which is retained and degraded in the ER, xcex11-ATHONG KONG, is affected by such inhibitors in that ER degradation is accelerated by glucosidase inhibitors and delayed by mannosidase inhibitors (Liu, Y., et al., J. Biol. Chem. (1997) 272:7946-7951; Liu, Y., et al., J. Biol. Chem. (1999) 274:5861-5867).
In general, it is understood that addition and trimming of oligosaccharide side chains are significant factors in the secretion, degradation, and transport of secretory, membrane and lyosomal glycoproteins. It is also known that transport of secretory and membrane glycoproteins from the ER to their appropriate destination depends on the interaction of the innermost glucose residue of oligosaccharide side chains with the ER molecular chaperones calnexin and calreticulin so that trimming of the oligosaccharide coupled to the asparagine residue in the peptide backbone by, for example, glucosidases I and II influence the proper folding and translocation of glycosylated proteins.
Experimental work related to the present invention has been described by applicants in Marcus, N.Y., and Perlmutter, D. H., Gastroenterology (2000) 118:1160 (meeting abstract entitled xe2x80x9cGlucosidase and Mannosidase Inhibitors Mediate Increased Secretion of A1 Antitrypsin Zxe2x80x9d; Burrows, J. A. J., et al., Proc. Natl. Acad. Sci (USA) (2000) 97:1796-1801 (article entitled xe2x80x9cChemical Chaperones Mediate Increased Secretion of Mutant alpha 1-Antitrypsin (alpha 1-AT) Z: A Potential Pharmacological Strategy for Prevention of Liver Injury and Emphysema in alpha 1-AT deficiencyxe2x80x9d); Marcus, N.Y., et al., J. Biol. Chem. (2000) 275:1987-1992 (article entitled xe2x80x9cGlucosidase and Mannosidase Inhibitors Mediate Increased Secretion of Mutant alpha 1-Antitrypsin Zxe2x80x9d); and Teckman, J. H., et al., Am. J. Physiol-Gastro and Liver Physiol. (2000) 278:G39-G48 (article entitled xe2x80x9cRole of Ubiquitin in Proteosome al Degradation of Mutant alpha 1-Antitrypsin Z in the Endoplasmic Reticulumxe2x80x9d). The contents of these publications are incorporated herein by reference.
It has now been found that inhibitors of glucosidase enhance the secretion of the mutant xcex11-ATZ glycoprotein without impairing its degradation in the ER. Inhibitors of mannosidase I also enhance secretion of mutant xcex11-ATZ, but delay its degradation in the ER. Compounds which are imino sugars or reduced forms thereof, such as derivatized deoxynojirimycin, are also useful in the methods of the invention.
Thus, in one aspect, the invention is directed to treat conditions associated with xcex11-antitrypsin deficiency caused by the presence of mutant xcex11-ATZ glycoprotein which method comprises administering to a subject in need of such treatment an effective amount of an inhibitor of glucosidase so as to enhance the secretion of the mutant xcex11-ATZ. In another aspect, the invention relates to methods to treat hepatotoxic conditions caused by the presence of mutant xcex11-ATZ in the endoplasmic reticulum which method comprises administering an effective amount of a glucosidase inhibitor to a subject in need of such treatment. In still another aspect, the invention is directed to ameliorating emphysema in individuals wherein the emphysema is caused by an xcex11-AT deficiency due to the presence of the mutant xcex11-ATZ which method comprises administering to individuals in need of such treatment an effective amount of a glucosidase inhibitor or a mannosidase I inhibitor.
Also useful in the various methods of the invention set forth above are imino sugars and their reduced forms. Imino sugars include analogs of hexoses or pentoses wherein the 5 or 4 position contains amino as opposed to hydroxy.
In other aspects, the invention relates to pharmaceutical compositions containing the aforementioned inhibitors and compounds.
Clinical studies have shown that only partial correction is needed for prevention of both liver and lung injury in patients having xcex11-antitrypsin deficiency. See Wu, Y., et al., Proc. Natl. Acad. Sci. (PNAS) U.S.A. (1994) 91:9014-9018; Campbell, E. J., et al., J. Clin. Invest. (1999)104:337-344). Thus, the ability of glucosidase inhibitors and mannosidase I inhibitors to enhance the secretion of xcex11-ATZ, which retains significant portion of the activity of the wild type xcex11-AT, make such compounds effective medications for treating the clinical symptoms associated with deficiency caused by the presence of xcex11-ATZ. Secretion can also be enhanced by 4-phenylbutyric acid (PBA).
Suitable subjects are those individuals whose genetic composition results in the production of the mutant form of xcex11-antitrypsin, xcex11-ATZ. These individuals can be determined by known methods, such as genetic typing and immunological tests. If subjects present with symptoms such as emphysema or liver malfunction, determination of whether or not the individual produces xcex11-ATZ is straightforward, routine, and readily performed. For use of the method of the invention in preventing the onset of such conditions, a suitable screening program would be desirable.
As used herein, xe2x80x9ctreatxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d includes ameliorating the effects of a condition already present as well as preventing the onset of symptomologies. Thus, xe2x80x9ctreatmentxe2x80x9d includes both therapeutic and prophylactic protocols.
The active ingredients in the compositions used in the methods of the invention are preferably glucosidase inhibitors, since these inhibitors both enhance the secretion of xcex11-ATZ and enhance its degradation in the ER. Such inhibitors include castanospermine (CST), which is a polyhydroxy alkaloid isolated from plant sources known to inhibit enzymatic glycoside hydrolysis, N-butyldeoxynojirimycin (BDNJ), N-nonyl DNJ (NDNJ), N-hexyl DNJ (HDNJ), and N-methyldeoxynojirimycin (MDNJ). These compounds are reduction products of imino sugars; it is understood that imino sugars and their reduction products are useful in the invention methods. Suitable mannosidase I inhibitors include deoxymannojirimycin (DMJ), which inhibits both mannosidase I and mannosidase II, and kifunensine (KIF), which inhibits mannosidase I. Other known inhibitors of glucosidase and mannosidase I can also be used.
Deoxynojirimycin is the reduction product of an imino sugar; an alternative name for deoxynojirimycin is 5-amino-1,5-dideoxy glucopyranose.
In addition to use of glucosidase or mannosidase inhibitors per se, it appears that, imino sugars or their reduction products generally are useful in the methods of the invention. Additional imino sugar compounds similar to CST are disclosed in Jacob, G. S., et al., Cur. Opin. Struct. Biol. (1995) 5:605-611; Winchester, B., et al., Glycobiol (1992) 2:199-210. While it appears that many imino sugars and their reduction products inhibit hydrolysis of glycose moieties, applicants wish not to be bound by any particular theory of the mechanism by which these imino sugars are able to exert their desirable effects on xcex11-ATZ retention in the ER.
The compositions of the invention are preferably administered systemically. For systemic use, the compounds herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal) or enteral (e.g., oral or rectal) delivery according to conventional methods. In general, because the disorders associated with the presence of xcex11-ATZ are chronic rather than acute, continued administration will generally be necessary. Therefore, preferably, administration will be by an enteral route, most preferably oral. Intravenous administration, less preferred, can be by a series of injections or by continuous infusion over an extended period. Administration by injection or other routes of discretely spaced administration can be performed at intervals ranging from weekly to once to three times daily. Alternatively, the compounds disclosed herein may be administered in a cyclical manner (administration of compound; followed by no administration; followed by administration of compound, and the like). Mixture of active compounds may also be used.
In general, pharmaceutical formulations will include a compound of the present invention in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, borate-buffered saline containing trace metals or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, lubricants, fillers, stabilizers, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington""s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton Pa., which is incorporated herein by reference. Pharmaceutical compositions for use within the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art. Various devices for metered drug delivery or sustained drug delivery may also be used.
Aqueous suspensions may contain the active ingredient in admixture with pharmacologically acceptable excipients, comprising suspending agents, such as methyl cellulose; and wetting agents, such as lecithin, lysolecithin or long-chain fatty alcohols. The said aqueous suspensions may also contain preservatives, coloring agents, flavoring agents, sweetening agents and the like in accordance with industry standards.
Parenteral preparations comprise particularly sterile or sterilized products. Injectable compositions may be provided containing the active compound and any of the well known injectable carriers. These may contain salts for regulating the osmotic pressure.
If desired, the compounds can be incorporated into liposomes by any of the reported methods of preparing liposomes for use in treating various conditions. The present compositions may utilize the compounds noted above incorporated in liposomes in order to direct these compounds to macrophages, monocytes, as well as other cells and tissues and organs which take up the liposomal composition. The liposome-incorporated compounds of the invention can be utilized by parenteral administration, to allow for the efficacious use of lower doses of the compounds. Ligands may also be incorporated to further focus the specificity of the liposomes.
Suitable conventional methods of liposome preparation include, but are not limited to, those disclosed by Bangham, A. D., et al., J. Mol. Biol. (1965) 23:238-252, Olson, F., et al., Biochim. Biophys. Acta. (1979) 557:9-23, Szoka, F., et al., PNAS (1978) 75:4194-4198, Kim, S., et al., Biochim. Biophys. Acta. (1983) 728:339:348, and Mayer, et al., Biochim. Biophys. Acta. (1986) 858:161-168.
As defined herein, an xe2x80x9ceffective amountxe2x80x9d of a composition is that amount which produces a statistically significant effect. For example, an xe2x80x9ceffective amountxe2x80x9d for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant decrease in symptoms of emphysema and/or hepatotoxicity. For use in prophylactic context, effective amounts are those which succeed in preventing the onset of symptoms or minimizing the severity of such symptoms. Such effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation, and the judgment of the practitioner and other factors evident to those skilled in the art. The dosage required for the compounds of the invention is manifested as an observed decrease in symptomology. General guidance for treatment regimens can also be obtained from experiments carried out in animal models of the disease of interest. See, also, Current Opinion in Structural Biology (1995) 5:605-611 for a review of studies on glycosylation inhibitors in biological systems.
Generally, the compounds of the invention can be administered to a typical human on a daily basis as an oral dose of about 1 mg/kg-1000 mg/kg, and more preferably from about 1 mg/kg to about 500 mg/kg. The parenteral dose will appropriately be 20-100% of the oral dose. While oral administration may be preferable in most instances (for reasons of ease, patient acceptability, and the like), alternative methods of administration may be appropriate for selected compounds and selected conditions.